title: Direct and simple detection of recombinant proteins from cell lysates using differential scanning fluorimetry.
abstract: A simple, inexpensive, and universal method to quantify the recombinant proteins in Escherichia coli cell lysate using differential scanning fluorimetry (DSF) is reported. This method is based on the precise correlation between Δ(fluorescence intensity) determined by DSF and the amount of protein in solution. We first demonstrated the effectiveness of the DSF method using two commercially available enzymes, α-amylase and cellobiase, and then confirmed its utility with two recombinant proteins, amylosucrase and maltogenic amylase, expressed in E. coli. The Δ(fluorescence intensity) in DSF analysis accurately correlated with the concentration of the purified enzymes as well as the recombinant proteins in E. coli cell lysates. The main advantage of this method over other techniques such as Western blotting, enzyme-linked immunosorbent assay (ELISA), and green fluorescence protein (GFP) fusion proteins is that intact recombinant protein can be quantified without the requirement of additional chemicals or modifications of the recombinant protein. This DSF assay can be performed using widely available equipment such as a real-time polymerase chain reaction (RT-PCR) instrument, microplates or microtubes, and fluorescent dye. This simple but powerful method can be easily applied in a wide range of research areas that require quantification of expressed recombinant proteins.
authors.affiliation: Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea.
authors.forename: Dong-Ho
authors.initials: DH
authors.lastname: Seo
authors.forename: Jong-Hyun
authors.initials: JH
authors.lastname: Jung
authors.forename: Hae-Yeong
authors.initials: HY
authors.lastname: Kim
authors.forename: Cheon-Seok
authors.initials: CS
authors.lastname: Park
mesh_terms: D002457:Cell Extracts / Q000737:chemistry*; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis*
chemical_list: D002457:Cell Extracts; D011994:Recombinant Proteins
keywords: Differential scanning fluorimetry; Recombinant protein; SYPRO orange dye